1. INTRODUCTION:

The cleaning processes used in pharmaceutical operations lead to consistent, validated manufacturing processes. Cleaning validation is performed once after a chromatographic method is developed (when using hplc) to identify the residue from various manufacturing instruments in the first place. The importance of cleaning method validation lies in the fact that it resists cross contamination and increases the sensitivity of the drug that is to be manufactured in the said pharmaceutical equipment’s^{. 1-7}. The residue is usually at a very low concentration, hence in order to remove that low level of residue, the method becomes all the more sensitive. There are two methods of sampling for cleaning method. One is direct sampling technique; another is indirect sampling technique.

In the following experiment, the direct sampling technique is used which is done by swab technique. The Food and Drug Administration (FDA) made an acceptance limit which should be based on risk associated with residues of determined products. Therapeutic doses, general limit (10 ppm) and toxicity of drugs are based on the calculation of Acceptable Limit of Residues and Maximum Allowable carryover (MAC).^8-13Megestrol acetate is a steroid derivative used as an agent to increase appetite and fight weight loss in anorexic patients due to cancer. It also treats patients with cachexia, suffering from AIDS and cancer.¹⁴ In the present work we have collected the residues of Megestrol acetate from various parts of manufacturing equipment and analyzed it in HPLC. This method would be helpful in pharmaceutical industry for determination of residues on the surface of manufacturing area and equipment to avoid any potential cross contamination.

2. EXPERIMENTAL:

2.a. Chemicals and Reagents:

Megestrol acetate API of batch number ADWS/0026, was provided by Celon Labs, Hyderabad, India. HPLC grade Methanol (Merck, India), Milli-Q Water (Millipore), HPLC grade Acetonitrile (Merck, India) and 0.45µ PVDF filters were used in the process.

2.b. Instruments and apparatus:

Waters Alliance HPLC system with PDA detector was used. Empower3 software was used for data integration.

2.c. Standards preparation:

2.c.i Diluent preparation: Methanol was used as a diluent.

2.c.ii Preparation of standard stock solution-1 (50ppm): 25.2mg of megestrol acetate standard (equivalent to 25mg of megestrol acetate) was weighed accurately and transferred into a 50ml volumetric flask. Volume was made up with diluent.

2.c.iii. Preparation of standard solution-1(5ppm): 1ml of 50ppm standard stock was poured into 10ml volumetric flask, diluted with diluent and made up to the mark and mixed well.

2.c.iv. Preparation of standard stock solution-2 (50ppm):25.2mg of megestrol acetate (equivalent to 25mg of megestrol acetate) standard was weighed accurately and transferred into a 50ml capacity flask. Volume was made up with diluent.

2.c.v. Preparation of standard solution-2(5ppm): 1ml of 50ppm standard stock was poured into 10ml volumetric flask, diluted with diluent and made up to the mark and mixed well.

2.c.vi. Preparation of Sample (Swab Sample): After collecting swab samples, they were transferred into a test tube containing 10ml of diluent. sonicated for 5 mins, then swab was pressed on the sides of test tube, removed and was used for injection in HPLC.

2.c.vii. Preparation of Sample (Rinse Sample) : Swab stick was taken and inserted into a test tube containing 10ml of diluent. According to swab technique swabbing is done on the stainless-steel plate for several times by using swab stick and dipped in between into test tube. Test tube was sonicated along with swab stick for 5 min. and the solution was taken into a vial as swab blank.

2.d. HPLC Method:

A HPLC method using primesil C18(4.6x150mm,5µm) column with isocratic elution was 25µl, the flow rate was 1ml/min, run time was 20 mins, both the column and sample temperature were fixed to ambient i.e., 25˚c and the sample was detected using a Spectro photometric UV (or) PDA detector at a wavelength of 280nm, three trials were employed in order to analyze the residue extracted in the swab, in which the mobile phase composed of water (A) and Acetonitrile (B) in % v/v in different proportions. The optimized composition was 65:35 (B: A) and held for 15 minutes. With this megestrol peak showed isocratic elution and the satisfying retention time (R_t) was found within 5 minutes, the chromatogram obtained is shown in Fig.1. So, after optimization 350:650%v/v (A: B) of mobile phase solution was prepared for further use. The method was validated for various parameters according to ICH Q2B guidelines.

2.e. Validation of Analytical method:

Validation was carried out to the optimized analytical method trial-3, according to ICH Q2 (R1) guidelines^10]. Parameters like System suitability test, Specificity, Precision, LOD and LOQ, Linearity, Accuracy and Robustness of the method were assessed.

3. RESULT AND DISCUSSION:

Validation of Analytical Method:

3.a System suitability test:

Blank, standard solution-1 in six replicates, standard solution-2 in duplicate, according to the test procedure injected into HPLC, and tested the parameters of suitability for the device. Results obtained are shown in Table 1.

Table 1: System suitability results

Parameter	Result
Tailing factor	0.93
% RSD	0.3
Similarity factor	0.99

Tailing factor, % RSD, Similarity factor are within the limits (Table 1) system meets the criteria of suitability for the method.

3.b. Specificity:

The RT of Blank and Swab Blank, of Megestrol Acetate should contain zero interference. Zero interference of the blank and swab blank at the retention time of analyte peak and peaks was obtained at 5.73 minute respectively. Hence method is specific.

3.c. Precision:

3.c.i System Precision

The % Relative Standard Deviation (% RSD) for the peak areas of injections of standard’s 6 replicas is NMT 5.0%. As the % RSD for peak areas of six identical injections of standard solution is within the limits (0.3%), system was found to be precise.

3.c.ii Method precision

Table:2. (a)Method Precision Results, (b) Intermediate Precision, (c) Comparative results of Method Precision and Intermediate Precision

(a)

Preparation	% Recovery of Megestrol acetate
Preparation	Method precision	Intermediate Precision
1	98.1	98.8
2	96.2	96.8
3	96.4	95.9
4	96.1	96.9
5	98.2	98.3
6	96.5	99.2
Cumulative Mean	97.3
Cumulative % RSD	1.2

(b)

Preparation	% Recovery
1	98.8
2	96.8
3	95.9
4	96.9
5	98.3
6	99.2
Mean	97.7
%RSD	1.3

(c)

Preparation No.	% Recovery
1	98.1
2	96.2
3	96.4
4	96.1
5	98.2
6	96.5
Mean Recovery	96.9%
%RSD (calculated)	1.0%
% RSD (acceptance criteria)	10.0%
% Recovery (accepted criteria)	80-120%

Each recovery should vary between 80.0% - 120.0%. The %RSD of six sample replicates should not be NMT 10.0.

As %RSD and % Recovery are within the acceptance criteria (Table 5), the method is found to be precise.

The individual recovery varying within 80.0% - 120.0%. The % RSD for % Assay of Megestrol acetate from 6 replicas of Intermediate Precision should be NMT 10.0. The cumulative % RSD for % Assay results of Megestrol acetate from twelve preparations of both Method and Intermediate Precision should be NMT 10.0.

As the individual % recovery, % RSD and overall % RSD are within the limits the method was found to be rugged.

3.d. Signal to Noise ratio:

The signal to noise proportion at LOD concentration should be about 3. The signal to noise proportion at LOQ concentration should be about 10. Signal to noise ratio at LOD and LOQ concentration are within the limits i.e. 3.4 and 10.46 respectively.

3.e. Linearity:

Analytical process linearity was achieved over the LOQ level range up to 300 percent of target concentration. Manifold solutions were prepared and injected in HPLC device over the concentration spectrum of LOQ level to 300 per cent covering six stages. Calculated the regression axis, slope, intercept and percent Y-Intercept correlation coefficient at 100 percent bias.

Table:3. (a) Linearity results of Megestrol Acetate (b) Precision results of Megestrol acetate at LOQ level and 300 % levels

(a)

Injection No.	Level
Injection No.	LOQ	300%
1	29147	19745332
2	29283	19649388
3	30361	19624277
4	29164	19655871
5	29572	19662067
6	29249	19652965
Mean	29463	19664983
STDEV	466	41453.8
% RSD	1.58	0.21

(b)

Level (%)	Megestrol Acetate
Level (%)	Concentration (ppm)	Area
LOQ	0.03	29463
50	2.52	3387827
100	5.04	6694818
150	7.56	9889130
200	10.07	13488835
300	15.11	19664983
Correlation Coefficient		0.9998

Regression coefficient		0.9996
Slope		1306246
Y-Intercept		79026.2
Y-Intercept at 100%		1.18
Residual sum of Squares		1.01871E+11

The correlation coefficient(r) is 0.9998 and is within the acceptance criteria (Table 2a).

Based on the above data (Table 2b), the Correlation Coefficient for Precision at LOQ level and 300% level results met the acceptance criteria. Hence the method is linear.

3.f. Accuracy:

Prepared and analyzed samples solutions at LOQ level, 50%, 100%, 150% and 300% of target concentrations and calculated the % individual recovery, % mean recovery and % RSD for Megestrol acetate at each level. Chromatograms obtained for sample from LOQ to 300% are shown in Figures 3.

Table 4: Accuracy Results

Preparation No.	Level (%)	Concentration (ppm)		% Individual recovery	Mean recovery	% RSD
		Added	Found
1	LOQ	0.03	0.03	100.8	102.8	1.8
2			0.031	103.5
3			0.031	104.2
1	50%	2.514	2.488	99	99.1	0.6
2			2.48	98.6
3			2.508	99.8
1	100%	5.021	4.826	96.1	96.9	1.2
2			4.931	98.2
3			4.845	96.5
1	200%	10.057	9.674	96.2	97.9	1.5
2			9.92	98.6
3			9.942	98.9
1	300%	15.086	15.023	99.6	99.8	0.2
2			15.043	99.7
3			15.094	100.1

Individual recovery should be ranging from 80 to 120%. For LOQ recovery should be between 70 to 130%. The % RSD for % Individual recovery at each level NMT 5.0. The % individual recovery, % mean recovery and % RSD for Megestrol acetate at each level met the acceptance criteria (Table 3). Hence the method is found to be accurate.

3.g. Range:

Based on Precision Linearity the range of method is 0.03ppm to 15.11ppm.

3.h. Robustness:

Table 5: (a) Effect of variation in flow rate, (b) Effect of variation in column temperature results(c) Effect of variation in organic phase ratio

(a)

Parameter	Low Flow	Actual Flow	High Flow	Acceptance criteria
Parameter	0.9 mL/min	1.0 mL/min	1.1 mL/min	Acceptance criteria
Main peak RT	6.37	5.73	5.21	---
Tailing Factor	0.91	0.93	0.92	NMT 2.0
% RSD	0.2	0.3	3.1	NMT 5.0
Similarity factor	0.99	0.99	1.01	0.95 - 1.05

(b)

Parameter	Low Temperature 20°C	Actual Temperature 25°C	High Temperature 30°C	Acceptance criteria
Main peak RT	5.94	5.73	5.53	---
Tailing Factor	0.92	0.93	0.93	NMT 2.0
% RSD	1.3	0.3	1.6	NMT 5.0
Similarity factor	1	0.99	0.99	0.95 - 1.05

(c)

Parameter	Low-organic	Actual	High- organic	Acceptance criteria
	Buffer: Acetonitrile 350:683 (% v/v)	Buffer: Acetonitrile 350:650 (% v/v)	Buffer: Acetonitrile 350:618 (% v/v)
	Buffer: Acetonitrile 350:683 (% v/v)	Buffer: Acetonitrile 350:650 (% v/v)	Buffer: Acetonitrile 350:618 (% v/v)
Main peak RT	5.38	5.41	4.98	---
Tailing Factor	1.05	1.05	1.06	NMT 2.0
% RSD	0.05	0.2	0.04	NMT 5.0
Similarity factor	0.98	0.98	0.98	0.95- 1.05

The above results (Table 4a) reveal that the method is robust over the range of flow rate from 0.9 mL/min to 1.1mL/min. System suitability parameters were analyzed as per the protocol by variation in column oven temperature at a low (20 °C) and a high temperature (35°C). Based on above results the method is found to be robust over the range of Column temperature 20 -30°C. The results reveal that the method is robust over the given range of organic phase variations in the mobile phase.

3.i. Solution stability:

Table 6: (a)Standard solution stability results, (b) Sample solution stability results, (c) Mobile phase stability results

(a)

Time	Initial	24 Hours		48 hours
Time	Initial	RT	2-8 °C	RT	2-8 °C
Similarity factor	NA	0.99	0.99	1.04	1.03

(b)

Time		Sample (% Recovery)	% Difference from Initial
Initial		98.1	NA
24 Hours	Room temperature	96.6	1.5
24 Hours	2-8°C	96	2.1
48 hours	Room temperature	101.2	-3.1
48 hours	2-8°C	100	-1.9

(c)

Parameter	Initial	Day 1	Day 2	Acceptance criteria
Main Peak RT	5.72	5.73	5.73	---
Tailing Factor	0.93	0.94	0.94	NMT 2.0
% RSD	0.3	0.1	0.2	NMT 5.0
Similarity factor	0.99	1	0.99	0.95 - 1.05

The similarity factor for Standard solution should be in between 0.95 to 1.05 at each interval. The sample solution was considered stable, if the difference in % recovery results from initial to 24 hours and 48 hours should be not more than ± 5.0 %. Based on the data (Table 5), the similarity factor for standard met its acceptance criteria up to 48 hrs. So, the standard solution and sample solution were stable for 48 hours on bench top as well as in refrigerator conditions. The difference in % recovery for sample met its acceptance criteria up to 48hrs.

Based on the results obtained (Table 5c) the mobile phase was found to be stable up to 48 hours on bench top condition.

4. CONCLUSION:

The cleaning method proposed in this article to quantify the residues of megestrol acetate collected from pharmaceutical manufacturing equipment was simple and economical as common reagents were used. The developed method was validated as per ICH Q2B guidelines. %RSD at each level was in between 80.0% to 120.0%, Correlation Coefficient and Precision at LOQ level and 300% level were found to be satisfactory. Robustness was carried out by varying flow rate, temperature and organic mobile phase in which the method was found robust all over the range. Solution stability was established over a range of 48 hours at room temperature and in refrigerator (2-8°C) conditions, results met the acceptance criteria and the solution was found to be stable for 48hrs in both the conditions. Based on the results obtained, the method was found to be specific, precise, rugged, robust, linear and accurate in the range of LOQ (0.03ppm) level to 300% (15 ppm) of residue concentration, thus the developed method for residue analysis is a sensitive analytical tool assuring the effectiveness of the method adopted.

5. ACKNOWLEDGEMENT:

The authors are thankful to Celon Laboratories Pvt. Ltd, Hyderabad-500081, Telangana, India and Department of Pharmaceutical Chemistry, Manipal College of Pharmaceutical Science, MAHE, Manipal-576104, Karnataka, India for providing facilities to perform this research work.

6. CONFLICTS OF INTEREST:

The authors declare that there is no conflict of interest.

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Received on 28.11.2022 Modified on 23.05.2023

Research J. Pharm. and Tech 2023; 16(12):6039-6044.

DOI: 10.52711/0974-360X.2023.00980